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Journal of Clinical Microbiology, March 2000, p. 1241-1243, Vol. 38, No. 3
Chugai Pharmaceutical Co. Ltd., Central
Research Laboratories, 3-41-8 Takada, Toshima-ku, Tokyo
171-8545,1 and National Institute of
Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo
162-8640,2 Japan
Received 5 August 1999/Returned for modification 3 November
1999/Accepted 17 December 1999
A new detection method for human parvovirus B19 DNA was established
using PCR coupled with a hybridization protection assay. The amplified
product was detected using acridinium ester-labeled DNA probes. By this
method, a few copies of B19 DNA were detected in human serum albumin.
Human parvovirus B19 (B19) is known
to cause erythema infectiosum (fifth disease) in children
(1) and chronic anemia in immunocompromised patients.
Recently, Saldanha and Minor (14) reported that blood
products from manufacturers around the world contained B19 DNA detected
by the PCR method. This report drew attention to the risk of infection
through blood products, especially in immunocompromised patients. Since
B19 is nonenveloped and thermostable even at 80°C (9),
inactivation of the virus is difficult if it is contained in a blood
product. Therefore, a sensitive and rapid method of detecting B19 in
blood products is required. We have attempted to establish a
hypersensitive detection method involving the use of PCR (13, 15,
20, 22) with a hybridization protection assay (HPA) (2,
12).
To estimate the number of B19 DNA copies per assay, about 4.7 kb of B19
DNA (nucleotides [nt] 215 to 4891) was amplified from serum of an
erythema infectiosum patient, cloned in plasmid pUC19, and propagated
in Escherichia coli. HB101. The B19 DNA fragment was excised
from the plasmid and purified. The concentration of the DNA was
determined by measuring A260. Three B19-positive
plasma samples were supplied by H. Sato, Fukuoka Red Cross Blood
Center. The concentrations of B19 DNA in plasma samples 1, 2 and 3 determined by the quantitative and competitive PCR method by H. Sato
were 1.34 × 1010, 2.13 × 109, and
2.60 × 1010 copies/ml, respectively.
Extraction of DNA from the plasma samples or human serum albumin (HSA)
solution was performed by using an SMI-Test Kit (Sumitomo Metal
Industries, Ltd., Tokyo, Japan) in accordance with the instructions supplied with the kit.
Two oligonucleotides primers were designed to amplify a 398-bp segment
in the VP1-VP2 region of B19 DNA (17, 20) and named PVVPa
3187(+) (5'-CAA AAG CAT GTG GAG TGA GG-3' [nt 3187 to 3206]) and
PVVPb 3584( The reaction mixture (100 µl) for PCR amplification consisted of 10 µl of 10× Gene Taq Universal Buffer, 0.2 mmol of deoxynucleoside triphosphates, 2.5 U of Taq polymerase (Wako Pure Chemicals
Industries, Ltd., Osaka, Japan), 15 pmol of each primer, and 10 µl of
the template. Ten microliters of silicone oil was added to the reaction mixture. Amplification was performed as follows: 94°C for 3 min, followed by 25 or 40 cycles of 94°C for 30 s, 52°C for
30 s, and 72°C for 1 min, and finally 72°C for 5 min using a
GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, Conn.).
For the HPA, two oligonucleotide probes were designed and named
PARV3313(+) (5'-CTG CCA CAA TGC CAG TGG AAA GGA GC-3') and PARV3348 (+)
(5'-GCA CCA TTA GTC CAA TAA TGG GAT AC-3'). Both probes were labeled
with acridinium ester (AE) as described by Arnold et al.
(3). Ten microliters of amplified PCR products was mixed
with 90 µl of TE buffer (10 mM Tris hydrochloride, 1 mM EDTA [pH
8.0]) and denatured by heating at 95°C for 5 min. The sample was
then chilled on ice for 5 min. Fifty microliters each of AE-labeled
probe and 100 µl of hybridization buffer were added, and the sample
was incubated at 60°C for 15 min. To achieve hydrolysis of
unhybridized probes, 300 µl of hydrolysis buffer was added and the
mixture was incubated at 60°C for 10 min. After 5 min at room
temperature, chemiluminescence signals from AE on hybridized probes
were measured using a Leader I Luminometer (Gen-Probe Inc., San Diego,
Calif.) and expressed as relative light units.
Ninety microliters of 5% HSA solution was spiked with 10 µl of a
diluted plasma sample which contained 1, 10, or 100 copies of B19 DNA.
DNA was extracted and served as the template for PCR. As shown in Fig.
1, the plasma sample containing a few
viral copies exhibited high numbers of relative light units after 40 cycles of amplification and HPA. With 25 cycles of amplification and HPA, results were rather unstable. We therefore adopted 40 cycles of
amplification for detection.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a Hypersensitive Detection Method
for Human Parvovirus B19 DNA
ABSTRACT
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) (5'-CTA CTA ACA TGC ATA GGC GC-3' [nt 3584 to 3565]).
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FIG. 1.
Detection of B19 DNA. B19 DNA was extracted from plasma
samples which were previously determined to have B19 DNA contamination
by quantitative and competitive PCR. PCR amplification was then
performed for either 25 or 40 cycles. The horizontal axis indicates the
B19 DNA concentration, and the vertical axis indicates
chemiluminescence; the axes are log based. Symbols: 
, specimen 1, 25 cycles; 
, specimen 2, 25 cycles; 
, specimen 3, 25 cycles; 
,
negative control, 25 cycles; 
, specimen 1, 40 cycles; 
, specimen
2, 40 cycles; 
, specimen 3, 40 cycles; 
, negative control, 40 cycles. RLUs, relative light units.
Neither human herpes simplex virus type 1, human cytomegalovirus (Sigma Chemical Co., St. Louis, Mo.), nor human hepatitis B virus (7) reacted with this system, which shows the specificity of the PCR-HPA method (data not shown).
To evaluate the variability of the method, spiking tests were performed
using seven different samples of HSA solution and a plasma solution
containing B19. The results showed that a single B19 DNA copy could be
detected using this method in all of the HSA solutions tested (Table
1).
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When the viral DNA was spiked into a 25% HSA solution, efficiency of DNA extraction was variable because of the high viscosity of the solution and assay results were not reproducible. When a 5% HSA solution was used, this variability was not observed (data not shown).
In this study, the primers were designed within the conserved VP1-VP2 region of the B19 genes based on the DNA sequence in the GenBank database (3, 4, 5, 6, 17, 18). The HPA probes were also placed in the well-conserved region among various B19 genes for maximum detection. The sensitivities of detection were quite similar for three plasma samples and one DNA clone. We expect that we could detect almost any B19 DNA by this method.
With this detection method, we could detect a few copies of B19 DNA in contaminated blood products in only 6 h. In comparison, Southern blotting analysis using a radioactive probe (8) or a chemiluminescent probe requires about 30 h to perform (11, 22). Recently, the Red Cross Blood Center of Japan introduced a receptor-mediated hemagglutination test for large-scale screening of infectious B19 virus in blood donation samples. The method takes only 3 h to perform, but its sensitivity is as low as 105 copies/ml (16, 19).
In conclusion, a new method for detecting B19 DNA has been established using PCR primer pairs and two newly synthesized probes for HPA. This method is specific and sensitive enough to detect a single copy of B19 DNA per assay in a 10-µl sample.
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ACKNOWLEDGMENTS |
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We thank H. Sato for supplying B19-positive plasma and Ruairi Mac Siomoin for his generous advice and help with English.
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FOOTNOTES |
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* Corresponding author. Mailing address: Chugai Pharmaceutical Co. Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171-8545, Japan. Phone: 81-3-3987-7196. Fax: 81-3-3989-0785. E-mail: satokza{at}chugai-pharm.co.jp.
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Journal of Clinical Microbiology, March 2000, p. 1241-1243, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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